Basement membranes (BMs) are organized extracellular structures which separate endothelial or epithelial cells from connective tissue. The long-range objectives are to elucidate the structure, assembly mechanism, and function of BMs, with particular reference to its heparan sulfate proteoglycan (HSPG) component. This information will then serve as the basis for the elucidation of BM alterations in pathological states such as diabetes, various blister-forming diseases, and invasion by metastatic cells. My approach to these problems makes use of a novel cell culture model system, the murine M1536-B3 cell, which produces isolatable cell-free BM Sacs, containing laminin, entactin, and HSPG. We have demonstrated that these cells produce two additional HSPGs which are localized in the medium and on the cell surface. We will ascertain the relationships between these three forms of HSPG through structural analyses and the isolation and characterization of cell mutants defective in HSPG formation. This information will be used to formulate a molecular mechanism by which HSPG are targeted to specific culture compartments. We will also determine structural features of BM HSPG which are required for their interaction with other BM components. The results of these studies will provide important new information on the role of HSPG in the structure, assembly, and function of BMs. The development of this BM-producing cell culture model system should also prove very useful in the study of the effects of hormones and therapeutic agents upon BM formation in disease states (e.g., diabetes) thus alleviating the use of many experimental animals for certain types of studies. (A)